The development of technologies related to biological sciences has led to a rapid increase in available genetic information. The completion of the sequencing of the human genome and the Human Genome Project's policy of instant data accessibility has created a fundament for studying gene-expression in physiological and pathological conditions. Techniques such as gene expression microarrays, differential display polymerase chain reaction (DD-PCR) and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) together with statistical clustering algorithms have provided tools for characterization of gene-expression patterns and pathways. This has led to the defining of new subcategories of known diseases, based on gene expression profiles, with different prognoses and potentially different responses to specific drugs and other treatment modalities.
DNA microarray technology has been the main contributor to the rapidly accumulating gene expression data. Using this technology, gene expression-based classifications have been developed for several malignant diseases. At present, gene expression microarray technology, however, has major technical shortcomings as a diagnostic method in a clinical laboratory as it is fairly insensitive and the quantitative precision is only moderate.
Gene expression analysis in a clinical setting is subjected to specific technical challenges due to the characteristics of the different types of clinical samples. Solid tissue samples such as histological paraffin-embedded or frozen tissue specimen usually contain a heterogeneous mixture of different cell types. Measurement of gene expression in a specific cell or tissue type within the sample requires isolation of cells or islets of cells from microscopic sections, e.g. by microdissection, or scoring of the relative amounts of different cell types present in sections cut from the sample. In needle biopsies and cytological samples the amount of sampled cells is usually small and the exact amount of cells is unknown. In blood samples the proportion of target cells is in many cases small and enrichment of specific cell types in the samples is required. Messenger RNA (mRNA) recovery from these types of samples is often limited, thus increasing the sensitivity requirements on the techniques used for gene expression analysis. In addition, sample-to-sample variations in mRNA degradation and recovery occur during sample preparation and storage as well as during nucleic acid purification. In order to obtain comparable results from tissue samples obtained in vivo, normalization for sample-to-sample variations in mRNA levels and integrity is required.
qRT-PCR has been widely used to validate results on gene expression levels that have been obtained using gene expression microarrays. The sensitivity of qRT-PCR is sufficient for quantitative measurement of gene expression in samples containing minimal amounts of cells or even single cells. Because of the logarithmic nature of the amplification of nucleic acids during PCR, this technique is sensitive to tube-to-tube variations due to small differences in reaction efficiencies. To overcome this problem, RNA or DNA internal control templates can be added to the samples to monitor the reaction efficiencies in individual reactions. Sample-to-sample variations in mRNA levels and integrity is typically controlled by normalizing mRNA expression levels of specific genes of interest against the expression levels of housekeeping genes or the amount of total RNA or ribosomal RNA (rRNA) in the sample, or by comparative quantification of mRNA from multiple genes in the same sample.
A method of modifying the size of the cDNA template during reverse transcription in order to discriminate it from genomic DNA has been described by Joo et al. (in Journal of Virological Methods 100 (2002) pages 71-81, and in patent application KR2002089746 A). In this technique a size-modifying-anchor primer is used in the reverse transcription reaction to insert a modified primer-binding site into the generated cDNA. This technique relies on the assumption that genomic DNA remains double-stranded when reverse transcription is performed under non-denaturing conditions, and thus cannot function as a template for reverse transcription. In the amplification step the cDNA and genomic DNA templates from the same gene are amplified using one common upstream primer and separate down-stream primers. The generated cDNA- and genomic DNA-derived amplicons differ in length to allow separate detection and quantification. Generally, RT-PCR techniques require amplification of a sequence that traverses at least one exon-exon boundary in order to enable separation of the cDNA- and genomic DNA-derived amplification products. The technique described by Joo et al. enables differentiation of cDNA from its corresponding genomic DNA within the boundaries of a single exon after RT-PCR amplification. It also allows comparison of relative levels of gene-specific mRNA and DNA.
The present invention provides a method for quantitative and/or comparative assessment of the relative amounts of mRNA transcripts present in a cell or tissue sample. In this method sequence-modified cDNA templates are competitively co-amplified with a reference DNA template using the very same primers. This enables using genomic DNA contained in, or added to the sample, as a universal reference template to normalize for tube-to-tube variations in amplification efficiencies between separate gene-specific amplification reactions. By quantitatively measuring the amounts and determining the relative levels of the amplification products derived from sequence-modified cDNA and reference DNA templates, a gene-specific cDNA over DNA ratio is obtained in each individual amplification reaction. By combining the cDNA over DNA ratios for each of the analyzed genes, a sample-specific gene-expression profile is generated, that reflects the relative amounts of mRNA transcripts originally present in the sample.
In the present invention, sequence-modified cDNA and reference DNA templates are co-amplified with the very same primers in the same reaction vessels. Thus, the relative levels of the cDNA and reference template derived amplification products remain constant even when amplification reactions are run to the plateau phase. This enables optimization of the sensitivity of the assay for samples containing only minimal amounts of mRNA transcripts.